Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.

The apoplast serves as the first battlefield between the plant hosts and invading microbes; therefore, work on plant-pathogen interactions has increasingly focused on apoplastic immunity. In the present study, we identified three proteins in the apoplast of cotton (Gossypium sp.) root cells during interaction of the plant with the fungal pathogen Verticillium dahliae. Among these proteins, cotton host cells secrete chitinase 28 (Chi28) and the cysteine-rich repeat protein 1 (CRR1), while the pathogen releases the protease VdSSEP1. Biochemical analysis demonstrated that VdSSEP1 hydrolyzed Chi28, but CRR1 protected Chi28 from cleavage by Verticillium dahliae secretory serine protease 1 (VdSSEP1). In accordance with the in vitro results, CRR1 interacted with Chi28 in yeast and plant cells and attenuated the observed decrease in Chi28 level that occurred in the apoplast of plant cells upon pathogen attack. Knockdown of CRR1 or Chi28 in cotton plants resulted in higher susceptibility to V. dahliae infection, and overexpression of CRR1 increased plant resistance to V. dahliae, the fungus Botrytis cinerea, and the oomycete Phytophthora parasitica var. nicotianae. By contrast, knockout of VdSSEP1 in V. dahliae destroyed the pathogenicity of this fungus. Together, our results provide compelling evidence for a multilayered interplay of factors in cotton apoplastic immunity.

Fertilizer application can increase yields, but nutrient runoff may cause environmental pollution and affect soil quality. A network-structured nanocomposite used as a soil conditioner is beneficial to crops and soil. However, the relationship between the soil conditioner and soil microbes is unclear. We evaluated the soil conditioner’s impact on nutrient loss, pepper growth, soil improvement, and, especially, microbial community structure. High-throughput sequencing was applied to study the microbial communities. The microbial community structures of the soil conditioner treatment and the CK were significantly different, including in diversity and richness. The predominant bacterial phyla were Pseudomonadota, Actinomycetota, and Bacteroidota. Acidobacteriota and Chloroflexi were found in significantly higher numbers in the soil conditioner treatment. Ascomycota was the dominant fungal phylum. The Mortierellomycota phylum was found in significantly lower numbers in the CK. The bacteria and fungi at the genus level were positively correlated with the available K, available N, and pH, but were negatively correlated with the available P. Our results showed that the loss of nutrients controlled by the soil conditioner increased available N, which improved soil properties. Therefore, the microorganisms in the improved soil were changed. This study provides a correlation between improvements in microorganisms and the network-structured soil conditioner, which can promote plant growth and soil improvement.